1. Field of the Invention
The present invention relates to matrices carrying simultaneously several different enzymatic functions and more particularly to complexes constituted by enzymes bound irreversibly on insoluble supports, as well as to the process for the preparation of these matrices. The present invention also relates to a process for the preparation, by means of said matrices, of oligonucleotides and polynucleotides with specific end groups.
2. Description of the Prior Art
It is known to fractionate polyribonucleotides by action of enzymes, to obtain various polynucleotides and oligonucleotides, and notably oligo U (or uridine-oligonucleotides) and oligo G (or guanosine-oligonucleotides). These enzymatic actions comprise essentially two stages, of which the first consists of subjecting the polyribonucleotides used to enzymatic hydrolysis by using either the ribonuclease of sheep's kidney [K. KASAI and M. GRUNBERG-MANAGO : Eur. J. Biochem. I (1967), 152], or beef pancreatic ribonuclease [G. SCHMIDT -- The nucleic Acids I (1955), p.555, Chargaff and Davidson Ed.] if it is sought to obtain uridine-oligonucleotides (that is to say oligonucleotides ended by a pyrimidine group), or ribonuclease T.sub.1 separated from takadiastase of Aspergillus orizae, if it is sought to obtain guanosine-oligonucleotides (that is to say oligonucleotides ended by a purine group) [Egami TAKAHASHI and UCHIDA - Progress in nucleic Acids and Research and Molecular Biology, XII, 1964, 59]. This enzymatic hydrolysis cleaves the polymeric chain at the 5'-phosphate junction, giving rise to a 3'-phosphate group, and it is followed by a second stage, which is a dephosphorylation by the alkaline phosphatase of Escherichia Coli.
The enzymes successively applied are in solution, so that it is necessary, at the end of the operations which have just been described, to separate the products obtained from the enzymes used, which are often very resistant so that these processes are difficult to apply and the yield of oligonucleotide obtained is very low.
To avoid these difficult separating processes which are complicated and expensive, it has been proposed to substitute for the enzymes in solution used in the course of the two stages of the process which has just been described, enzymes rendered insoluble by binding to a support. The work of PORATH and KRISTIANSEN ("The Proteins" vol. I, 3rd Ed., Academic Press) has, in fact, made known enzymes rendered insoluble by binding on a matrix of glass or of agarose. However, the processes proposed, which successively apply insoluble enzymes, if they resolve in part the difficulties of separation of the soluble enzymes, also however involve a considerable number of operations, so that it is still necessary, in any case, to resort to processes involving long and complicated operations, which are of little practicality on the industrial scale.